Enhancers of Cellular Cannibalism for Use to Sensitize Tumors to Radiation Therapy

ABSTRACT

The present invention is drawn to the use of the compounds highlighted in Tables 1 &amp; 2 and analogs thereof, for enhancing IR-mediated cellular cannibalism in cancer cells. Said compounds are herein called “enhancers of IR-mediated cellular cannibalism”. They can be used to enhance tumor immunogenicity and/or to induce a significant protective anticancer immune response in subjects that will receive or that have received a radiotherapy treatment. In other words, said compounds can be used to potentiate a radiotherapy treatment in a subject in need thereof. Said compounds are preferably chosen in the group consisting of: Mebhydroline 1,5-napthalene disulfonate salt, Flurbiprofen, Minaprine dihydrochloride, Myricetin, Digoxin, Digitoxin, Lanatoside, LOPA87, VP331, RN-1-026, SG6163F, VP450, and VP43.

SUMMARY

The present invention is drawn to the use of the compounds highlightedin Tables 1 & 2 and analogs thereof, for enhancing IR-mediated cellularcannibalism in cancer cells. Said compounds are herein called “enhancersof IR-mediated cellular cannibalism”. They can be used to enhance tumorimmunogenicity and/or to induce a significant protective anticancerimmune response in subjects that will receive or that have received aradiotherapy treatment. In other words, said compounds can be used topotentiate a radiotherapy treatment in a subject in need thereof. Saidcompounds are preferably chosen in the group consisting of: Mebhydroline1,5-napthalene disulfonate salt, Flurbiprofen, Minaprinedihydrochloride, Myricetin, Digoxin, Digitoxin, Lanatoside, LOPA87,VP331, RN-1-026, SG6163F, VP450, and VP43.

BACKGROUND OF THE INVENTION

Radiotherapy is one of the most common anti-tumor strategies used incancer treatments. More than half of patients with cancers are treatedwith radiotherapy. The use of radiotherapy (alone or in combination withsurgery and chemotherapy) is central to the management of tumors of headand neck, breast, lung, prostate, digestive and cervical cancer¹.Exposure to ionizing radiation (IR) of tumor tissues triggers variouslethal processes such as apoptosis, autophagic cell death, mitoticcatastrophe or senescence.

These mechanisms contribute to the destruction of cancer cells in theirradiated tissues, but can also induce the immunogenicity of irradiatedtumor cells^(2, 3), modify the tumor microenvironment and allow thedevelopment of an anti-tumor immune response involving the release ofdanger signals (adenosine triphosphate and HMGB1 protein)^(4, 5), theactivation of purinergic receptors (notably P2X7 and P2Y2)⁴, the TLR4receptor^(2, 3) and the NLRP3 inflammasome⁴. The relationship betweenthe local tumor response and the stimulation of the immune system isillustrated by describing a remote effect on non-irradiated cancercells. This paradoxical effect, also known as the abscopal effect, hasbeen studied for many years in different murine tumor models as well asin patients⁶⁻⁹. Its detection correlates positively with the presence ofa high amount of proinflammatory cytokines (such as TNF alpha), ofinterferon (IFN) gamma producing T cells and requires the development ofan effective immunological response to be observed^(10, 11). This effectis also observed in patients treated with radiotherapy in combinationwith immuno-modulating agents (such as Ipilimumab or Interleukin-2)¹².The description of the abscopal effect reinforces the recentdescriptions in many animal models of a major contribution of variouscomponents of the immune system to the response to radiotherapy. Thecentral role of immune cells (including tumor-associated macrophages, Tlymphoid cells and dendritic cells) has recently been studied andhighlighted by the influence of their therapeutic modulation withmodulators of immunological checkpoints (such as the use of monoclonalantibodies against CTLA4 or PD1) or macrophage activation (withmonoclonal antibodies directed against CSF1R). Despite the central roleof radiotherapy in the anti-tumor therapeutic arsenal, the biologicalprocesses involved in the elimination of tumor cells are still partiallydefined and controversial. Early studies on radio-induced tumor celldeath are relatively old and occurred before the recent discovery of newcell death processes as well as of the central role of the immune systemin the elimination of tumor cells following irradiation.

Scientific and technological advances in recent years revealed theexistence of several processes of cell death. A classification of celldeath modalities mainly built on morphological and functional criteriahas been proposed¹³ and subdivided lethal processes into three distincttypes: type I cell death (also known as apoptosis), type II cell death(or autophagy) and type III cell death (or necrosis). Initiallyorganized on the apparent distinction between type I and III cell deathmodalities, between processes that are regulated oraccidentally-induced, or by distinguishing cell deaths that areassociated with the induction of by-stander inflammatory response invivo, to lethal processes that were thinking silent or tolerogenic, thisclassification that still misnamed the type II cell death as autophagy(which is mainly an intracellular survival mechanisms required for themaintenance of cellular homeostasis) did not reflect the aptitude ofnecrotic deaths to be genetically controlled or immunogenic as revealedby the recent molecular characterization of necroptosis^(14, 15). Inaddition, cell death subroutines that did not or partially reveal thesestereotyped morphological, metabolic and biochemical modifications (suchas mitotic death and cornification) have been less studied and weregrouped in a poorly defined subgroup of cell death modalities, alsoknown as atypical cell death subgroup¹³.

In recent years, several new cell death mechanisms (such as entosis oremperitosis) have been described and associated to this neglectedsubgroup of cell death modalities^(16, 17). The characterization ofthese atypical death modalities highlighted the existence of cell deathprocesses that are not achieved like typical cell death modalities, in acell autonomous manner, but are elicited after the engulfment of livedcells by neighbor cells.

During decades, non-cell autonomous deaths (NCADs) have beenepisodically observed and studied. Initially referred asphagoptosis^(18, 19) or emperipolesis²⁰, these processes have beeninvolved in the control of erythrocyte, neutrophil, platelet or T cellhomeostasis^(18, 21, 22) and thus, proposed as major physiological formsof cell death in the body¹⁸. Detected as cell-in-cell structures, NCADshave been also observed during ex vivo culture of primary cells²³ orduring histological analyses of physiological processes (such as embryoimplantation²⁴ and intrahepatic depletion of autoreactive T cells²⁵) orhuman diseases (including cancer²³, inflammatory syndromes²³, duringinfectious diseases^(26, 27)). Mainly detected after homotypicinteractions between malignant cells in human tumors (including breast,cervical and colon carcinomas or melanomas), NCADs are also detectedafter heterotypic interactions between tumor cells and stroma cells²⁸,tumor cells and immune effectors (such as lymphocytes²⁹ and NK cells³⁰),but may also be observed after interactions between immune effectors andepithelial cells (as revealed by the destruction of T cells in thymicnurse cells³¹ or autoreactive T cells in liver²⁵). Recently, NCAD wasalso associated with infectious diseases. The engulfment of Humanimmunodeficiency virus 1 (HIV-1) or Epstein barr virus (EBV) infectedcells by uninfected cells has been detected during in vitro co-cultureand the degradation of internalized infected cells proposed as the firststep of a new cell-to-cell mode of viral transmission between infectedcells and host cells²⁶ ²⁷, underlining the ability of NCADs to alsocontribute to microbial pathogenesis.

The first step of non-cell autonomous death programs starts with theinteraction of two cellular partners through membrane adhesion receptors(such as E- or P-cadherins) or stress receptors (such as lipoproteinreceptor-related protein) and is followed by the formation of adherentjunctions between interacting cells that activates signaling pathways onboth interacting cells and may involve small GTPases (such as Rho³² andCdc42³³) and ROCK kinases¹⁶. Then, the modulation of actomyosincontractibility and the reorganization of the actin cytoskeleton on thelevel of “target” cells were shown to favor their invasion into hostcells^(32, 34). This process is distinct from cellular cannibalism thatcan also trigger NCADs through the activation of specific signalingpathways (such as phagocytosis-related signaling pathways that involvecytoskeleton remodeling cell division cycle 42 (CDC42), chemokine (C—X—Cmotif) ligand 1 (CXCL1) or chemokine (C—X—C motif) ligand 6 (CXCL6)) onhost cells and leads to an active engulfment of target cells³³. Onceinternalized, engulfed cells may be targeted by “host” lysosomal enzymes(such as cathepsins and granzymes) and eliminated through distinctlethal mechanisms that may involve several modulators of typical celldeaths (such as cytochrome c, caspases or autophagy-related (ATG)proteins). Entosis, a non-cell autonomous death, initially describedafter homotypic interactions between breast cancer cells, iscell-in-cell invasion mechanism that does not require the activation ofcaspases to eliminate engulfed cells¹⁶. Inversely the engulfment ofnatural killer (NK) by tumor cells initiates emperitosis, a progammedcell-in-cell death that requires caspase 3 activation and DNAfragmentation³⁰, revealing that engulfed cells can be eliminated throughcaspase-dependent or caspase independent processes.

Despite considerable efforts, the approaches developed over the last 10years to enhance the effectiveness of radiotherapy have not met theexpected clinical success. Among the reasons for the failures of theprevious approaches, it can be pointed out that most of the approachesdeveloped so far have characteristics different from those of thecurrent project. Moreover, these approaches have not been developedusing rational approaches based on the characterization of themechanisms of cell death and resistance to therapy.

Specifically, they never took into account the impact of the immunesystem in the elimination of tumor cells. Finally, they merelytransposed in radiotherapy applications coming from the field of medicaloncology without taking into account specific mechanisms of cell deathafter irradiation.

In this context, there is a need to identify and characterize what arethe modulators of cell death after radiotherapy irradiation and evaluatewhat are the immunological consequences of administering thesemodulators in cancer patients.

DESCRIPTION OF THE INVENTION

Despite the intensive biological and pharmaceutical research implementedto better characterize cellular and biochemical processes associatedwith anticancer treatments, lethal mechanisms responsible for thetherapeutic effects of radiotherapy, which is one of the most frequentanticancer treatment used in clinic, are still unknown. Lethal processes(such as apoptosis and mitotic catastrophe) that have been detected inresponse to ionizing radiation were not directly implicated in treatmentefficiency³⁵, suggesting that additional cell death modalities that arestill unknown may contribute to therapeutic effects of radiotherapy.

The present project is based on the discovery of a new cell deathmechanism (the cellular cannibalism) that has never been detected byothers after RI or other anticancer treatments. Usually, in tumorvaccination experiments, cancer cells were exposed to cytotoxicanticancer treatment in vitro until 70% of the cells exposephosphatidylserine on the outer leaflet of the plasma membrane⁴⁸.Interestingly, the present results reveal that chemical compoundsselected on the basis of their ability to enhance IR-mediated cellularcannibalism (IRCCE) convert cancer cells into a vaccine that stimulateantitumor immune responses. More specifically, the present resultsreveal for the first time that the combination of IRCCE+IR elicit anIR-mediated anticancer immune response in absence of a significantincrease of death of treated and irradiated cells (FIG. 5A), suggestingthat cellular cannibalism or cellular cannibalism-associated signalingpathways contributes to the induction of tumor immunogenicity afterirradiation.

Irradiation is commonly used for treating cancer. Often combined withchemotherapy and/or surgery, irradiation therapy encompasses both localand total body administration as well as a number of new advances,including radio-immunotherapy. The cytotoxic effect of irradiation onneoplastic cells arises from the ability of irradiation to cause a breakin one or both strands of the DNA molecule inside the cells. Cells inall phases of the cell cycle are susceptible to this effect. However,the DNA damage is more likely to be lethal in cancerous cells becausethey are less capable of repairing DNA damage. Healthy cells, withfunctioning cell cycle checkpoint proteins and repair enzymes are farmore likely to be able to repair the radiation damage and functionnormally after treatment. Nevertheless, irradiation brings side effectsthat are a burden for the patients. The side effects of irradiation aresimilar to those of chemotherapy and arise for the same reason, i.e.,the damage of healthy tissues. Irradiation is usually more localizedthan chemotherapy, but this treatment is still accompanied by damages topreviously healthy tissue. Many of the side effects are unpleasant, andirradiation also shares with chemotherapy the disadvantage of beingmutagenic, carcinogenic and teratogenic in its own right. While normalcells usually begin to recover from treatment within two hours oftreatment, mutations may be induced in the genes of the healthy cells.These risks are elevated in certain tissues, such as those in thereproductive system. Also, it has been found that different peopletolerate irradiation differently. Doses that may not lead to new cancersin one individual may in fact spawn additional cancers in anotherindividual. This could be due to pre-existing mutations in cell cyclecheckpoint proteins or repair enzymes, but current practice would not beable to predict at what dose a particular individual is at risk. Commonside effects of irradiation include bladder irritation, fatigue,diarrhea, low blood counts, mouth irritation, taste alteration, loss ofappetite, alopecia, skin irritation, change in pulmonary function,enteritis, sleep disorders, and others.

It would be highly advantageous to provide patients with a potentiatingagent that triggers a synergistic effect with irradiation, therebyallowing the patient to be less exposed to irradiation therapy. Thiswould indeed reduce the above-mentioned side-effects, while achieving animproved beneficial result. This is also an aspect of the presentinvention.

Altogether, it is proposed that, given the highlighted importance ofcellular cannibalism on the elimination of irradiated cancer cells andthe increasing importance of the immune response in tumor response toirradiation, cellular cannibalism is a “desirable” death that should befavored during cancer treatments in order to efficiently eliminateirradiated cancer cells. This can be achieved by administering to thepatients cellular cannibalism enhancers as demonstrated by the presentinventors in the experimental part below.

Definitions

The term “irradiation therapy” is commonly used in the art to refer tomultiple types of radiation therapy including internal and externalradiation therapy, radio-immunotherapy, and the use of various types ofradiation including X-rays, gamma rays, alpha particles, beta particles,photons, electrons, neutrons, radioisotopes, and other forms of ionizingradiation. As used herein, the terms “irradiation therapy”, “radiationtherapy”, “radiation” and “irradiation” are inclusive of all of thesetypes of radiation therapy, unless otherwise specified. There aredifferent types of radiotherapy machines, which work in slightlydifferent ways. The number and duration of the radiotherapy sessionsdepend on the type of cancer and where it is located in the body. Asuperficial skin cancer may need only a few short treatments, whereas acancer deeper in the body may need more prolonged treatment.

The terms “suppressing tumor growth”, “treating tumor growth”, and“treating cancer”, and the like refer to reducing the rate of growth ofa tumor, halting tumor growth completely, causing a regression in thesize of an existing tumor, eradicating an existing tumor and/orpreventing the occurrence of additional tumors upon treatment with thecompositions, kits or methods of the present disclosure. “Suppressing”tumor cell growth means any or all of the following states: slowing,delaying, and stopping tumor growth, as well as tumor shrinkage.“Delaying development” of a tumor means to defer, hinder, slow, retard,stabilize, and/or postpone development of the disease. This delay can beof varying lengths of time, depending on the history of the diseaseand/or individual being treated. Tumor cell growth can be assessed byany means known in the art, including, but not limited to, measuringtumor size, determining whether tumor cells are proliferating using a³H-thymidine incorporation assay, or counting tumor cells.

As used herein, “synergy” or “synergistic effect” when referring tocombination administration of a compound of the present disclosure inconjunction with radiation means that the effect of the combination ismore than additive when compared to administration of the compound(s)and radiation alone.

“Potentiate” means, in the context of this application to enhance orincrease the effect of, for example, a radiotherapy treatment, or topromote or strengthen, for example, a biochemical or physiologicalaction or effect.

“Effective amount” refers to an amount of a compound as described hereinthat may be therapeutically effective to treat a cancer associated withthe instant disclosure. The precise amount of these compounds requiredwill vary with the particular compounds or derivatives employed, the ageand condition of the subject to be treated, and the nature and severityof the condition. However, the effective amount may be determined by oneof ordinary skill in the art with only routine experimentation. Aneffective amount of radiation can be determined without undueexperimentation by one of ordinary skill in the art. Radiationparameters, such as dosing amount and frequency are well-known in theart.

“Pharmaceutically acceptable” refers to those compounds, materials,compositions, and/or dosage forms which are, within the scope of soundmedical judgment, suitable for contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem complications commensurate with a reasonablebenefit/risk ratio.

“Pharmaceutically acceptable salts” refer to derivatives of thedisclosed compounds wherein the parent compound is modified by makingacid or base salts thereof. The compounds of this disclosure form acidand base addition salts with a wide variety of organic and inorganicacids and bases and includes the physiologically acceptable salts whichare often used in pharmaceutical chemistry. Such salts are also part ofthis disclosure. Typical inorganic acids used to form such salts includehydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, phosphoric,hypophosphoric and the like. Salts derived from organic acids, such asaliphatic mono and dicarboxylic acids, phenyl substituted alkonic acids,hydroxyalkanoic and hydroxyalkandioic acids, aromatic acids, aliphaticand aromatic sulfonic acids, may also be used. Such pharmaceuticallyacceptable salts thus include acetate, phenylacetate, trifluoroacetate,acrylate, ascorbate, benzoate, chlorobenzoatc, dinitrobenzoate,hydroxybenzoatc, mcthoxybenzoate, methyl benzoate, o-acetoxybenzoate,naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate,β-hydroxybutyrate, butyne-1,4-dioate, hexyne-1,4-dioate, cabrate,caprylate, chloride, cinnamate, citrate, formate, fumarate, glycollate,heptanoate, hippurate, lactate, malatc, maleate, hydroxymaleate,malonate, mandelate, mesylate, nicotinate, isonicotinate, nitrate,oxalate, phthalate, teraphthalate, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, propiolate,propionate, phenylpropionate, salicylate, sebacate, succinate, suberate,sulfate, bisulfate, pyrosulfate, sulfite, bisulfite, sulfonate,benzene-sulfonate, p-bromobcnzenesulfonate, chlorobenzenesulfonate,ethanesulfonate, 2-hydroxyethanesulfonate, methanesulfonate,naphthalene-1-sulfonate, naphthalene-2-sulfonate, p-toleunesulfonate,xylenesulfonate, tartarale, and the like. Bases commonly used forformation of salts include ammonium hydroxide and alkali and alkalineearth metal hydroxides, carbonates, as well as aliphatic and primary,secondary and tertiary amines, aliphatic diamines. Bases especiallyuseful in the preparation of addition salts include sodium hydroxide,potassium hydroxide, ammonium hydroxide, potassium carbonate,methylamine, diethylaminc, and ethylene diamine.

Alternative embodiments provide a method of potentiating radiotherapycancer treatment as described, comprising the administration of a“slow-release formulation” that is able to release the active ingredientto the surroundings in a controlled, non-instant manner, time-dependentmanner. This slow-release formulation may comprise a biodegradablepolymer, for example selected from the group consisting of a homopolymerof lactic acid; a homopolymer of glycolic acid; a copolymer ofpoly-D,L,-lactic acid and glycolic acid; a water-insoluble peptide saltof a luteinizing hormone-releasing hormone (LHRH) analogue; apoly(phosphoester); a bis(p-carboxyphenoxy)propane (CPP) with sebacicacid copolymer; a polyanhydrides polymer;poly(lactide)-co-glycolide)polyethylene glycol copolymers; and anethylene-vinyl acetate copolymer.

In some specific embodiments of the method, the slow-release formulationreleases the IRCCE drug over a period of four or more weeks,alternatively over a period of one week or more, or alternatively over aperiod of few hours or more.

The methods, compositions formulations and uses described herein aresuitable for both humans and animals, preferably mammals.

Thus, as used herein, the term “subject” relates to any mammal includinga mouse, rat, pig, monkey and horse. In a specific embodiment, it refersto a human. A “subject in need thereof” or a “patient” in the context ofthe present invention is intended to include any subject that willbenefit or that is likely to benefit from the methods and pharmaceuticalcompositions of the present invention. The subject may suffer from acancer of any stage such that it could be an early non-invasive canceror could be a late stage cancer that has already progressed to formmetastases in the body.

“Patient” herein refers to animals, including mammals, preferablyhumans.

As used herein the term “cancer” is intended to include any form ofcancer or tumors. Non-limiting examples of cancers include brain cancer(e.g., glioma), gastric cancer, head-and-neck cancer, pancreatic cancer,non-small cell lung cancer, small cell lung cancer, prostate cancer,colon cancer, non-Hodgkin's lymphoma, sarcoma, testicular cancer, acutenon-lymphocytic leukemia and breast cancer. In a particular embodiment,the brain cancer is an astrocytoma, and more particularly, isglioblastoma multiforme; the lung cancer is either a small cell lungcarcinoma or a non small cell lung carcinoma; and the head-and-neckcancer is squamous cell carcinoma or adenocarcinoma.

The present invention is based on the discovery that known and unknowndrugs are able to enhance IR-mediated cellular cannibalism in vitro andin vivo in tumor-bearing animals.

As used herein, the terms “cellular cannibalism inducing drugs”,“cellular cannibalism inducing agents”, “cellular cannibalism inducingcompounds” and “cellular cannibalism enhancers” designate compounds thatare able to enhance the capacity of irradiation to trigger cellularcannibalism. Under these terms are designate compounds that are able toenhance IR-mediated cellular cannibalism (hereafter called IRCCE). Thechemical structures of the selected cellular cannibalism modulators arepresented in Table 1 and Table 2.

TABLE 1 Molecular weight Molecules Structures (g/mol) Myricetin

318.24 Minaprine dihydrochloride

371.3 Lanatoside C

985.12 Flurbiprofen

244.26 Mebhydroline 1,5- naphtalene- disulfonate

420.52 Azaguanine-8

152.11 Digitoxigenin

390.51 Doxorubicin hydrochloride

579.98 Digoxin

780.949

TABLE 2 Molecular Molecules Structures weight (g/mol) RN-1-001

320 VP43

425 VP450

287 SG6163F

348 RN-1-026

346 VP331

350 LOPA87

291

In total, the present inventors discovered 16 efficient cellularcannibalism enhancer compounds, namely: 8-Azaguanine, Mebhydroline1,5-napthalene disulfonate salt, Flurbiprofen, Minaprinedihydrochloride, Myricetin, Digoxin, Digitoxin, Lanatoside, Doxorubicinehydrochloride, LOPA87, VP331, RN-1-026, SG6163F, VP450, VP43. A numberof interesting compounds have been also identified. They are forexamples analogs of LOPA87 (LOPA90, LOPA93, LOPA 94, LOPA 101, LOPA104,LOPA105, LOPA106) and analogs of SG6143F (SG6144, SG6146 and SG6149).Therefore, in a preferred embodiment, the cellular cannibalism enhancersof the invention are chosen in the group consisting of: 8-Azaguanine,Mebhydroline 1,5-napthalene disulfonate salt, Flurbiprofen, Minaprinedihydrochloride, Myricetin, Digoxin, Digitoxin, Lanatoside, Doxorubicinehydrochloride, LOPA87, VP331, RN-1-026, SG6163F, VP450, and VP43, thestructures of which are displayed in Tables 1 and 2. In a more preferredembodiment, the cellular cannibalism enhancers of the invention arechosen in the group consisting of: Mebhydroline 1,5-napthalenedisulfonate salt, Flurbiprofen, Minaprine dihydrochloride, Myricetin,Digoxin, Digitoxin, Lanatoside, LOPA87, VP331, RN-1-026, SG6163F, VP450,and VP43. In an even more preferred embodiment, the cellular cannibalismenhancers of the invention are chosen in the group consisting of:8-Azaguanine, Minaprine dihydrochloride, LOPA87, VP331, and SG6163F.

They can also be analogs of LOPA87, such as LOPA90, LOPA93, LOPA 94,LOPA 101, LOPA104, LOPA105, and LOPA106.

They can also be analogs of SG6163F, such as SG6144, SG6146 and SG6149.

8-Azaguanine (C₄H₄N₆O, Purine analogue). The 8-Azaguanine is a purineanalogue with potential antineoplastic activity. 8-Azaguanine interfereswith the modification of transfer ribonucleic acid (tRNA) by competingwith guanine for incorporation into tRNA catalyzed by the enzymetRNA-guanine ribosyltransferase (tRNA-guanine transglycosylase). Alteredguanine modification of tRNA has been implicated in cellulardifferentiation and neoplastic transformation. 8-Azaguanine alsoinhibits the formation of 43S and 80S initiation complexes, therebyinterfering with initiation of translation and inhibiting proteinsynthesis. This agent inhibits tumor cell growth and stimulates celldifferentiation.

Mebhydroline 1,5-naphtalene disulfonate salt (C₁₉H₂₀N₂._(1/2)C₁₀H₈O₆S₂,a H1 histamine receptor antagonist). The Mebhydrolin (napadisylate) isan antihistamine, used for symptomatic relief of allergic symptomscaused by histamine release, including nasal allergies and allergicdermatosis.

Flurbiprofen (C₁₅H₁₃FO₂, phenylalkanoic acid derivative family ofnon-steroidal anti-inflammatory drugs (NSAIDs)). Flurbiprofen is used torelieve pain, tenderness, swelling, and stiffness caused byosteoarthritis (arthritis caused by a breakdown of the lining of thejoints) and rheumatoid arthritis (arthritis caused by swelling of thelining of the joints). Flurbiprofen is in a class of medications calledNSAIAs (nonsteroidal anti-inflammatory agents). It works by stopping thebody's production of a substance that causes pain, fever, andinflammation. This nonsteroidal anti-inflammatory agent of the propionicacid class is structurally and pharmacologically related to fenoprofen,ibuprofen, and ketoprofen, and has similar pharmacological actions toother prototypica NSAIAs. Flurbiprofen exhibits anti-inflammatory,analgesic, and antipyretic activities. The commercially availableflurbiprofen is a racemic mixture of (+)S- and (−) R-enantiomers. TheS-enantiomer appears to possess most of the anti-inflammatory, whileboth enantiomers may possess analgesic activity. Similarly to otherNSAIAs, the anti-inflammatory effect of flurbiprofen occurs viareversible inhibition of cyclooxygenase (COX), the enzyme responsiblefor the conversion of arachidonic acid to prostaglandin G2 (PGG2) andPGG2 to prostaglandin H2 (PGH2) in the prostaglandin synthesis pathway.This effectively decreases the concentration of prostaglandins involvedin inflammation, pain, swelling and fever. Flurbiprofen is anon-selective COX inhibitor and inhibits the activity of both COX-1 and-2. It is also one of the most potent NSAIAs in terms of prostaglandininhibitory activity.

Minaprine dihydrochloride (C₁₇H₂₂N₄O, amino-phenylpyridazineantidepressant). Minaprine dihydrochloride is a psychotropic drug whichhas proved to be effective in the treatment of various depressivestates. Like most antidepressants minaprine antagonizes behavioraldespair. Minaprine is an amino-phenylpyridazine antidepressant reportedto be relatively free of cardiotoxicity, drowsiness, and weight gain.More specifically, minaprine is an amino-phenylpyridazine antidepressantreported to be relatively free of cardiotoxicity, drowsiness, and weightgain. Similar to other antidepressant treatments, minaprine attenuatesthe beta-adrenergic receptor function. Studies have also shown thatminaprine improves memory consolidation and that repeated drugadministration leads to potentiation of this effect. Moreover, theeffects of minaprine on memory consolidation are related to itsdopaminergic action. Minaprine binds to serotonin type 2 receptors andto dopamine D1 and D2 type receptors. It also binds to the serotoninreuptake pump. Therefore, minaprine blocks the reuptake of both dopamineand serotonin. It is also, to a slight degree, cholinomimetic. Thus itmay exhibit both mood-brightening and nootropic properties. It also actsas a reversible inhibitor of MAO-A (RIMA). It has also been found toinhibit acetylcholinesterase.

Myricetin (C₁₅H₁₀O₈, benzopyrans, small molecule). ThePhosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gammaisoform has been proposed as cellular target.

Digoxin (C₄₁H₆₄O₁₄, Cardiac glycoside). Digoxin, a cardiac glycosidesimilar to digitoxin, is used to treat congestive heart failure andsupraventricular arrhythmias due to reentry mechanisms, and to controlventricular rate in the treatment of chronic atrial fibrillation.

Digitoxin (C₄₁H₆₄O₁₃, Cardiac glycoside). Digitoxin is a lipid solublecardiac glycoside that inhibits the plasma membrane sodium potassiumATPase, leading to increased intracellular sodium and calcium levels anddecreased intracellular potassium levels. In studies increasedintracellular calcium precedes cell death and decreased intracellularpotassium increase caspase activation and DNA fragmentation, causingapoptosis and inhibition of cancer cell growth.

Lanatoside C (C₄₉H₇₆O₂₀, Cardiac glycoside). Lanatoside C, isolated fromvarious species of digitalis, is used in the treatment of congestiveheart failure and cardiac arrhythmia.

Doxorubicine hydrochloride (C₂₇H₂₉NO₁₁, Cancer chemotherapy).Doxorubicin Hydrochloride is the hydrochloride salt of doxorubicin, ananthracycline antibiotic with antineoplastic activity. Doxorubicin,isolated from the bacterium Streptomyces peucetius var. caesius, is thehydroxylated congener of daunorubicin. Doxorubicin intercalates betweenbase pairs in the DNA helix, thereby preventing DNA replication andultimately inhibiting protein synthesis. Additionally, doxorubicininhibits topoisomerase II which results in an increased and stabilizedcleavable enzyme-DNA linked complex during DNA replication andsubsequently prevents the ligation of the nucleotide strand afterdouble-strand breakage. Doxorubicin also forms oxygen free radicalsresulting in cytotoxicity secondary to lipid peroxidation of cellmembrane lipids; the formation of oxygen free radicals also contributesto the toxicity of the anthracycline antibiotics, namely the cardiac andcutaneous vascular effects.

Chemical Compounds Obtained from the Screened Library

LOPA87, VP331, RN-1026, SG6163F, VP450 and VP43 have been obtained fromthe CEA library. 7 analogs of LOPA87 (LOPA90, LOPA93, LOPA 94, LOPA 101,LOPA104, LOPA105, LOPA106) and 3 analogs of SG6163F (SG6144, SG6146 andSG6149) were also identified.

Aspects of the Invention

In a first aspect, the present invention is drawn to the in vitro use ofthe above-identified compounds (highlighted in Tables 1 & 2) forenhancing IR-mediated cellular cannibalism (IRCCE) of cancer cells.

This aspect of the invention is performed in vitro. As disclosed herein,the terms “in vitro” and “ex vivo” are equivalent and refer to studiesor experiments that are conducted using biological components (e.g.cells or population of cells) that have been isolated from their usualhost organisms (e.g. animals or humans). Such isolated cells can befurther purified, cultured or directly analyzed to assess the presenceof the mutant proteins. These experiments can be for example reduced topractice in laboratory materials such as tubes, flasks, wells,eppendorfs, etc. In contrast, the term “in vivo” refers to studies thatare conducted on whole living organisms.

IR-mediated cellular cannibalism can be assessed by any conventionalmeans. In particular, this activity can be assessed by studying thecells under the microscope (by immunofluorescence orimmunohistochemistry) and by visually detecting cell engulfment eventsor cell-in-cell systems. Alternatively, it can be assessed by measuring,in said cell, the expression level of a protein selected from the groupconsisting of: p53, p53β, p53γ, and N-terminal isoforms of p53 that lackthe N-terminal transactivating domain, such as Δ40TP53 and Δ133TP53, orby measuring the expression level or the activity of the purinergic P2Y2receptor, and/or by measuring the extracellular ATP secreted by saidcells, as disclosed in WO2014/006227 which is incorporated herein byreference.

The compounds of the invention can also be used in vivo.

In this case, the compounds of the invention are preferably administeredto the patients in a pharmaceutical composition further containingpharmaceutically acceptable adjuvants. The “pharmaceutical compositionof the invention” therefore contains an efficient amount of at least oneof the compound of the invention (see Table 1 & 2 above), or analogsthereof, and pharmaceutically acceptable adjuvants or carriers. Examplesof non-aqueous adjuvants are propylene glycol, polyethylene glycol,vegetable oil and injectable organic esters such as ethyloleate. Otherpharmaceutically acceptable adjuvants include aqueous solutions andnon-toxic excipients including salts, preservatives, buffers and thelike. Intravenous vehicles include fluid and nutrient replenishers. Thecompositions may also include preservatives such as antimicrobialagents, anti-oxidants, chelating agents and inert gases. The pH andexact concentration of the various components of the pharmaceuticalcomposition can be adjusted according to well-known parameters.

The in vivo administration of the compounds of the invention (or of thecomposition of the invention) is intended to enhance the tumorimmunogenicity in subjects that will or that have undergone aradiotherapy treatment. While not being bound to any particular theory,it is believed that these compounds work synergistically with radiationtherapy by favoring cellular cannibalism and thereby the exposure ofparticular epitopes that induce a significant protective anticancerimmune response.

The present disclosure targets the use of the compounds disclosed inTables 1 & 2 above or their analogs for manufacturing a pharmaceuticalcomposition intended to be administered prior or after radiotherapy to asubject in need thereof.

It is also drawn to the use of the compounds disclosed in Tables 1 & 2above or their analogs for manufacturing a pharmaceutical compositionintended to potentiate a radiotherapy treatment to a subject in needthereof.

It is finally drawn to the use of the compounds disclosed in Tables 1 &2 above or their analogs for manufacturing a pharmaceutical compositionintended to treat cancer, in conjunction with radiotherapy, in a subjectin need thereof.

In a second aspect, the present invention is drawn to the compoundsdisclosed in Tables 1 & 2 above or their analogs, or any pharmaceuticalcomposition containing same,

-   -   for their use for enhancing IR-mediated cellular cannibalism in        patients suffering from cancer,    -   for their use for enhancing tumor immunogenicity in subjects        that will or that have received a radiotherapy treatment,    -   for their use for inducing a significant protective anticancer        immune response in subjects that will or that have received a        radiotherapy treatment,    -   for their use for potentiating a radiotherapy treatment in a        subject in need thereof,    -   for their use for treating cancer, in conjunction with        radiotherapy, in a subject in need thereof.

Importantly, administering the compositions of the invention will enableto reduce the dose of irradiation and therefore results in thealleviation of the side effects incurred by the radiation treatment.

The pharmaceutical compositions of the present invention can beadministered in the form of injectable compositions (e.g.,intravenously, intramuscularly, subcutaneously and intra-articularly),either as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid prior to injection also may beprepared. These preparations also may be emulsified.

Said pharmaceutical compositions can also be administered by otherroutes such as orally, nasally, rectally, topically, intravenously,intramuscularly, subcutaneously, sublingually, intrathecally,intraperitoneally, intra-articularly or intradermally. Preferably, theroute of administration is intravenously, intra-arterially or orally.Preferably, the pharmaceutical compositions are administeredintravenously or intra-arterially.

In specific embodiments, when the composition of the present inventionis for oral administration, the composition of the invention is in atablet, a solution or capsule such as a soft gel capsule for example. Inother specific embodiments, when the composition of the presentinvention is for oral administration, it has an enteric coating. Inother specific embodiments, when the composition of the presentinvention is for oral administration, it is an oil-based syrup.

In a particular embodiment, the compounds of the invention areadministered in a slow-release formulation.

The compositions of the present invention are administered in amountsand at frequencies sufficient to treat cancer, in conjunction with aradiotherapy treatment. A subject's progress can be determined bymeasuring and observing changes in the concentration of cancer markers;by measuring the actual size of the tumor over time and/or bydetermining any other relevant clinical markers which are well-known inthe art. The determination, measurement, and evaluation of suchcharacteristics and markers associated with clinical progress arewell-known to those of ordinary skill in the art.

In an embodiment, the compositions of the present invention areadministered prior to radiotherapy. The time of administration willdepend on the specific formulation and on the time necessary for thecellular cannibalism enhancer to reach the target cells. The time ofadministration will be chosen so as to provide the optimal concentrationof cellular cannibalism enhancer at the time of irradiation.

In another embodiment, the compositions of the present invention areadministered after the radiotherapy treatment has been applied to thepatient.

In another embodiment, the administration of the compositions of thepresent invention and the radiotherapy treatment has been appliedconcomitantly to the patient

The present inventors observed that the administration of minaprinedihydrochloride alone has intrinsic protective effects against tumordevelopment (see FIGS. 6B, 6G, 6L, 6P and 6Q). Consequently, minaprinedihydrochloride does not need to be combined with a radiotherapytreatment, it is efficient on its own. This is a surprising result,since minaprine dihydrochloride is known as an anti-depressant drug, andno anti-tumoral effect has ever been reported for this drug.

In a third aspect, the present invention therefore addresses minaprinedihydrochloride, or any pharmaceutical composition containing same,

-   -   for its use for treating patients suffering from cancer,    -   for its use for inducing a significant protective anticancer        immune response in cancer patients.    -   For its use for treating cancer when combined with a        radiotherapy or chemotherapy or immunotherapy treatment.

All the embodiments disclosed above for the pharmaceutical compositionsare transposable to this particular use.

Methods of Treatment

The present disclosure also provides methods aiming at enhancingIR-mediated cellular cannibalism (IRCCE) in patients suffering fromcancer, enhancing tumor immunogenicity in subjects that will or thathave received a radiotherapy treatment, inducing a significantprotective anticancer immune response in subjects that will or that havereceived a radiotherapy treatment, potentiating a radiotherapy treatmentin a subject in need thereof, and/or treating cancer, in conjunctionwith radiotherapy, in a subject in need thereof.

In these methods, the compounds of the invention (see Table 1, Table 2,and analogs thereof as detailed above) or the pharmaceuticalcompositions of the invention (as disclosed above) are administered tosaid subject prior to, or after or concomitantly the treatment withradiotherapy.

The present invention also targets a method for treating cancerpatients, comprising administering to said patients an effective amountof minaprine dihydrochloride, or any pharmaceutical compositioncontaining same, As a matter of fact, said anti-depressant drug has beenshown by the inventors to have a significant protective anticancerimmune response on its own.

Screening Methods

In another embodiment, the present invention relates to an in vitromethod of identifying a compound that may potentiate a radiotherapytreatment in a subject, said method comprising the steps of:

(a) adding a candidate compound to a tumor cell culture,

(b) treating said tumor cell culture with a radiation dose of 1-20 Gray,

(c) measuring the irradiation-mediated cellular cannibalism occurring insaid cell culture,

wherein a compound that enhances irradiation-mediated cellularcannibalism as compared to control levels will be able to potentiate aradiotherapy treatment.

As mentioned above, IR-mediated cellular cannibalism can be assessed byany conventional means. In particular, this activity can be assessed bystudying the cells under the microscope (by immunofluorescence orimmunohistochemistry) and by visually detecting cell engulfment eventsor cell-in-cell systems. Alternatively, it can be assessed by measuring,in said cell, the expression level of a protein selected from the groupconsisting of: p53, p53β, p53γ, and N-terminal isoforms of p53 that lackthe N-terminal transactivating domain, such as Δ40TP53 and Δ133TP53, orby measuring the expression level or the activity of the purinergic P2Y2receptor, and/or by measuring the extracellular ATP secreted by saidcells, as disclosed in WO2014/006227 which is incorporated herein byreference.

Alternatively, the treatment of the cell culture with irradiation can beperformed prior to the adding of the candidate compound to the cellculture.

In a preferred embodiment, the radiation dose used in step b) iscomprised between 1-10Gray, typically at 8Gray.

In a preferred embodiment, the tumor cell culture is treated with thecandidate compound for at least 12 hours, more preferably for at least20 hours, even more preferably for 24 hours, prior or after theirradiation occurs.

Said tumor cell culture can be a culture of HCT116 cells, of CT26carcinoma cells, of MCA205 fibrosarcoma cells.

The confirmation of the potentiating effect of the candidate compoundcan be performed as described in the examples below, i.e., by treatingthe appropriate tumor cell cultures with the compounds for at least 12hours, and applying an irradiation treatment, and then inject thesecells into immunocompetent mice, in order to confirm that a protectiveimmune response has been elicited.

According to the experimental part below, the compounds disclosed inTables 1 & 2 above and their analogs are herein designated as examplesof “enhancers of IR-mediated cellular cannibalism”.

FIGURE LEGENDS

FIG. 1 Cell Death Profiling by quantitative imaging flow-cytometry. (A)Principle of cell death profiling by quantitative flow imaging. (B)Validation of multiparametric and simultaneous detection of cell deathmodalities by quantitative imaging flow-cytometry induced bygamma-irradiation. Before co-culture, treated HCT116 cells andnon-treated HCT116 cells were respectively labeled with CMFDA(green/light grey) or CMTMR (red/dark grey) fluorescent vital probes.After 24 hours of co-culture, HCT116 cells were analysed for non-cellautonomous death (NCAD) (by detecting engulfment of CMTMR- orCMFDA-labeled HCT116 cells), for phosphatidylserine (PS) exposure (usingBiotin-AnnexinV and BV786-Streptavidin), for loss of plasma integrity(by following with DRAQ7 uptake) and for DNA content (using Hoechst33342). Using quantitative flow-cytmotery, the simultaneous detection ofNCA deaths and of typical cell deaths (Type I, II and III) on bothnon-treated and treated HCT116 cells will determine the cell deathprofiling obtained after cancer treatment. Representative images areshown (scale, 20 μm).

FIG. 2 Detection of γ-irradiation-elicited cell autonomous deathmodalities by quantitative imaging flow-cytometry. (A-E) Detection andquantification of plasma membrane integrity loss and PS exposureobserved after 24 hour co-culture of untreated (red/dark grey)CMTMR-labeled HCT116 cells and untreated (green/light grey)CMFDA-labeled HCT116 cells (A, C-E), of untreated (red/dark grey)CMTMR-labeled HCT116 cells and untreated (green/light grey)CMFDA-labeled HCT116 cells that have been extemporally mixed (C-E) orafter 24 hour co-culture of untreated (red/dark grey) CMTM-labeledHCT116 cells with (green/light grey) CMFDA-labeled HCT116 cells (green)that have been irradiated with 4 grays of y-ionizing radiation (B-E).The co-cultures have been performed in presence or absence of theindicated pharmacological death effector inhibitors. The detection ofplasma membrane integrity (with DRAQ7) and PS exposure (withBV786-streptavidin/Annexin V biotin) have been analyzed for theuntreated (red/dark grey) CMTMR⁺ HCT116 cells, for untreated(green/light grey) CMFDA⁺ HCT116 cells, for treated (green/light grey)CMFDA⁺ HCT116 cells and for total cell population (CMTMR⁺ or CMFDA⁺HCT116 cells). Representative dot plots (A,B) and quantitative data(C-E) are shown (means±SEM, n=3). (F,G) Representative cell cycledistributions of untreated (red/dark grey) CMTMR⁺ HCT116 cells,untreated (green/light grey) CMFDA⁺ HCT116 cells, treated (green/lightgrey) CMFDA⁺ HCT116 cells and for total cell population (CMTMR⁺ orCMFDA⁺ HCT116 cells) obtained after 24 hour co-culture of untreated(red/dark grey) CMTMR-labeled HCT116 cells with untreated (green/lightgrey) CMFDA-labeled HCT116 cells (F, H-J), of untreated (red/dark grey)CMTMR-labeled HCT116 cells and untreated (green/light grey)CMFDA-labeled HCT116 cells that have been extemporally mixed (H-J) orafter 24 hour co-culture of untreated (red/dark grey) CMTM-labeledHCT116 cells with (green/light grey) CMFDA-labeled HCT116 cells(green/light grey) that have been irradiated with 4 grays of γ-ionizingradiation (G-J) are shown. Quantitative data of cell cycle analysis areshown in (H-J) (means±SEM, n=3). * or # or $ represents p<0.05, ## or $$p<0.01, *** or ### p<0.001.

FIG. 3 Detection of γ-irradiation-elicited non-cell autonomous deathmodalities by quantitative imaging flow-cytometry and confocalfluorescence microscopy. (A-G) Cell-in-cell structures and target celldegradation were determined by quantitative imaging (A-C) and confocalfluorescent microscopy (D-G) after 24 hour co-culture of untreated(red/dark grey) CMTM-labeled HCT116 cells with (green/light grey)CMFDA-labeled HCT116 cells that have been irradiated with 4 grays ofγ-ionizing radiation (A), coculture of untreated (red/dark grey)CMTMR-labeled HCT116 cells with untreated (green/light grey)CMFDA-labeled HCT116 cells (B, C) or on CMTMR labeled (red/dark grey)HCT116 cells and untreated (green/light grey) CMFDA-labeled HCT116 cellsthat have been extemporally mixed (B, C). As previously described, cellshave been sequentially labeled after co-cultures with specificfluorescent probes like BV786-streptavidin-Annexin V biotin, DRAQ7, andHoechst 33342. Then, (red/dark grey) CMTMR-labeled HCT116 cellsinternalizing (green/light grey) CMFDA-labeled HCT116 cells (notedR(G)), and (green/light grey) CMFDA-labeled HCT116 cells internalizing(red/dark grey) CMTMR-labeled HCT116 cells (noted G(R)) were detected.Representative images are shown in (A) (scale, 20 m). Frequencies ofCell-in-Cell structures (B) and target cell degradation (C) are reported(means±SEM, n=3). Representative confocal images of cell-in-cellstructures (white arrow) (D) and target cell degradation (white dottedarrow) (E) detected during co-culture of untreated (red) CMTMR-labeledHCT116 cells with untreated or γ-irradiated (green) CMFDA-labeled cellsare shown (scale bar=10 m). (F-G) Frequencies of cell-in-cell structuresshowing (red/dark grey) CMTMR-labeled cells internalizing (green/lightgrey) CMFDA-labeled cells (noted R(G)), and (green/light grey)CMFDA-labeled cells internalizing (red/dark grey) CMTMR-labeled cells(noted G(R)) (F) and target cell degradation (G) have been determined(means±SEM, n=3); # or $ represents p<0.05, ## or $$ p<0.01, *** or $$$or ### p<0.001.

FIG. 4 Identification of cannibalism modulators triggered by ionizingradiation. Compounds from the Prestwick (A) and CEA (F) libraries weretested for their capacities to induce cellular cannibalism afterionizing radiation. Cannibal cells were detected after homotypiccultures of 8 gray γ-irradiation HCT116 cells in the presence or absenceof μM of the Prestwick (A) and CEA (E) library compounds. Each dotrepresents one compound. Representative images are shown in (B, G). (C,H) HCT116 cells were treated with the indicated drugs. After 24 h oftreatment, cell death was monitored by staining with 3,3dihexyloxacarbocyanine iodide (DiOC₆(3)) and PI, and the percentage ofdying (DiOC₆(3)^(low) PI⁻, open bars) and dead (DiOC₆(3)^(low) PI⁺,closed bars) cells was determined by cytofluorometry. Validation of thecannibalism inducers by fluorescent microscopy. Representative images(D, I) and quantification (E, J) are shown. Results are means±s.e.m. oftriplicate determinations.

FIG. 5. Identification of cannibalism modulators as immunogenic celldeath inducers. MCA205 (A) or CT26 (E, I, M, Q) cells were treated withthe indicated drugs. After 24 h of coculture with the indicatedtreatment, cell death was monitored by staining DiOC₆(3) and PI, and thepercentage of dying (DiOC₆(3)^(low) PI⁻, open bars) and dead(DiOC₆(3)^(low) PI⁺, closed bars) cells was determined bycytofluorometry. (B-D) MCA205 cells or (F-H, J-L, N-P, R-T) CT26 cellscocultured after x irradiation with the indicated compounds wereinoculated subcutaneously into the right flank of C56BL/6 or BALB/cmice, respectively. Seven days later, the mice were rechallenged withlive cells injected into the opposite flank, and tumor growth wasmonitored (five mice per group).

FIG. 6. Tumor vaccination experiments. CT26 cells were treated for 24hours with 10 μM VP331 (A, F, K), 10 μM Minaprine (B, G, L), 10 μMLopa87 (C, H, M), 10 μM SG6163F (D, I, N), 10 μM Azaguanine-8 (8-aza)(E, J, O) alone or combined with 8 Gy ionizing radiation and inoculated(s.c.) into immunocompetent BALB/c mice, which were rechallenged at theopposite flank 7 days later with the same cancer cells. The percentageof tumor-free mice was evaluated three times a week for the following 38days. Percentage of total tumor free mice (A-E), tumor free-mice at thefirst injection site (F to J, P) or at the second injection site (K toO, Q) are shown. (*P<0.05, **P<0.01, ***P<0.001, two-way ANOVA).

EXAMPLES

I. Material and Methods

Chemicals, Cell Lines and Culture Conditions

Unless otherwise indicated, chemicals were purchased from Sigma-Aldrich.Antibiotics, media, supplements for cell culture were obtained from LifeTechnologies. Benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone(Z-VAD-fmk) was from Bachem and recombinant mouse TNF-alpha from R&Dsystems. Human colon carcinoma HCT116 cells were cultured in McCoy's 5Amedium and murine fibrosarcoma cell line L929 in Dulbecco's modifiedEagle's medium. All the media were supplemented with 10%heat-inactivated fetal bovine serum (FBS), 10 mM HEPES buffers, 2 mML-glutamine, 10 U/mL penicillin sodium and 10 μg/mL streptomycinsulfate.

Irradiation

Cells were seeded in 6-well plates, 12-well plates or 25 cm² flasks andirradiated at indicated dose with gamma-ray irradiator IBL-637 (Cs¹³⁷, 1Gy/min, gamma CIS-Biolnternational, IBA, Saclay, France).

CellTracker™ Fluorescent Probes Labeling

Upon the removal of the culture medium, HCT116 cells were incubated withpre-warmed medium containing 10 μM of 5-chloromethylfluoresceindiacetate (CMFDA, green fluorescence) or5-(and-6)-(((4-Chloromethyl)Benzoyl)Amino)Tetramethylrhodamine (CMTMR,red fluorescence) (Molecular Probes-Life Technologie) for 45 min at 37°C. Thereafter, HCT116 cells were rinsed twice with pre-warmed medium,and incubated for 1 hour at 37° C. Stained cells were treated asindicated and cultured for cell death profiling analysis.

Cell Death Profiling by Quantitative Flow Imaging

Untreated HCT116 cells were labeled with CMFDA (green fluorescence,CMFDA⁺) or CMTMR, (red fluorescence, CMTMR⁺) and treated HCT116 cellswith CMFDA (green fluorescence, CMFDA⁺). The following cell mixtureswere performed: untreated CMTMR⁺ HCT116 cells were mixed with untreatedCMFDA⁺ HCT116 cells, or untreated CMTMR⁺ HCT116 cells were mixed withtreated CMFDA⁺ HCT116 cells. Then, cells were co-cultured during 24hours in the presence or absence of the pharmacological inhibitor ofROCK, Y27632 (30 μM), the pan-caspase inhibitor, Z-VAD-fmk (ZVAD, 100μM), the inhibitor of caspase-1, Ac-YVAD-cmk (YVAD, 100 μM), thenecroptosis inhibitor, Necrostatin-1 (NEC 1, 30 μM), the inhibitor ofthe vacuolar type H(+)-ATPase (V-ATPase) inhibiting autophagy,Bafilomycin A1 (BafA1, 50 nM), the inhibitor of Cdks with ananti-mitotic activity, Roscovitine (Rosco, 10 μM). After 24 hours ofco-culture, both detached and adherent cells were collected and stainedwith Hoechst 33345 (10 g/mL) during 1 hour at 37° C. in warmed completemedium. To detect phosphatidylserine (PS) exposure and plasma membranepermeability, labeled HCT116 cells were successively incubated withBiotin-AnnexinV (BD Pharmingen) as recommended by manufacturer, 0.5 gBV786-Streptavidin (BD Biosciences) and 3 M DRAQ7 (BioStatus) during 15minutes at 25° C. After washing with PBS solution, samples wereimmediately analyzed using an imaging flow cytometer FlowSight® (Amnis®,part of EMD Millipore). Data were acquired at a 20× magnification, usingINSPIRE software. The 405 nm, 488 nm, and 561 nm lasers were used forexcitation. Brightfield, Annexin V-BV786, DRAQ7, CMFDA, CMTMR andHoechst 33345 stainings were detected using respectively channels for420-480 nm, 745-800 nm, 642-745 nm, 480-560 nm, 595-642 nm and 430-505nm. At least 1000 events of cells per sample were analyzed. Additionalsingle-labeled controls were prepared to normalize fluorescent signalacross different channels. Acquired data were analyzed using the IDEASanalysis software (v6.1; Merck-Millipore). Gating strategy was thefollowing. Cells were gated for focused cells using the Gradient RMSfeature. Cells were gated for single cells using the aspect ratio andarea features. For the cannibalism detection, cells were gated in thedouble positive CMFDA⁺ and CMTMR⁺ staining.

Flow Cytometry and Confocal Fluorescent Microscopy

To detect PS exposure, plasma membrane permeability and cell cycleprogression, cells were after co-culture sequentially labeled withspecific fluorescent probes (such as FITC-conjugated AnnexinV, propidiumiodide, and Hoechst 33342) and analyzed by flow cytometry. Both detachedand adherent cells were collected and stained with Hoechst 33345 (10ug/ml) during 1 hour at 37° C. in warmed complete medium. After washingwith PBS, HCT116 cells were suspended in 1X binding buffer supplementedwith fluorescein isothiocyanate (FITC)-conjugated Annexin V (BDBiosciences) and propidium iodide (PI, 1 μg/mL) (Sigma), as permanufacturer's instructions. Samples were then analyzed using LSRII flowcytometer (Becton Dickinson) and the FlowJo software v10. For confocalfluorescence microscopy, HCT116 cells were fixed after co-culture in3,7% paraformaldehyde-PBS for 15 minutes and counterstained with 1 μg/mLHoechst 33342 (Invitrogen) for 15 minutes. Then, cells were analyzed byconfocal SPE microscope equipped with Apochromat 63x 1.3 NA and 63x 1.15NA oil immersion objectives. The Leica Aplication Suite (LAS) softwarewas used (Leica Microsystems).

Western Blots

Total cellular proteins were extracted in lysis buffer (containing 1%NP40, 20 mmol/L HEPES, 10 mmol/L KCl, 1 mmol/L EDTA, 10% glycerol,protease and phosphatase inhibitor tablets). Protein extracts (30 μg)were run on 4-12% NuPAGE® Novex® Bis-Tris gels (Life Technologies) andtransferred at 4° C. onto Immobilon polyvinyldifluoride (PVDF) membranes(Thermo Scientific). After blocking, membranes were incubated at 4° C.overnight with primary antibodies specific for: caspase-3 (#9662),cleaved caspase-3 (Asp175) (#9661), Myosin Light Chain 2 (MLC2) (#3672),phospho-MLC2 (Serl9) (#3675), LC3 A/B (#4108), p-(S)-CDKs Substrate(#9477) were obtained from Cell Signaling Technology. Antibodies againstGAPDH (#MAB374) were purchased from Millipore. Horseradishperoxidase-conjugated goat anti-mouse or anti-rabbit (SouthernBiotechnology) antibodies were then incubated during 1 h and revealedwith the SuperSignal West Pico® reagent (Thermo Fisher Scientific) orthe ECL™ Prime Western Blotting Detection System (GE Healthcare) usingan ImageQuant LAS 4000 software-assisted imager (GE Healthcare).

Statistical Analyses

Each experiment has been repeated at least three times, yieldingcomparable results. Unless otherwise indicated, figures illustratequantitative data from one representative experiment (means±SEM, n=3).Data were analyzed by means of Prism v. 5.03 (GraphPad Software, LaJolla, Calif., USA). Statistical significance was assessed by two-tailedStudent's t tests. In all experiments, p values <0.05 were considered asstatistically significant.

II. Results

Cell Death Profiling Analysis Using Multispectral Imaging Flow-CytometryAllows the Simultaneous Detection of Non-Cell Autonomous and CellAutonomous Death Modalities.

The ability of cells to die through NCA processes led us to reconsiderfrom a conceptual point of view our methodological approach to considercell death processes. Indeed, the choice of the morphological and/orbiochemical parameters to be considered as well as the technologicalapproach to be used to detect cell death predefines in advance theresults to be expected and does not allow or very rarely to identify newlethal processes such as cellular cannibalism or entosis. The field ofradiotherapy is also facing this problem. Indeed, it has been revealedin separate studies that irradiation can trigger many lethal processessuch as apoptosis, autophagic cell death, necrosis or mitoticdeath^(35,36). It has recently been shown in separate studies thatirradiation of the same cell type with the same doses could triggerapoptosis³⁷, but also mitotic death³⁸. In previous studies it wasrevealed that the onset of apoptosis and mitotic death observed veryrapidly following irradiation do not correlate with clonogenic survivalobserved in the longer term³⁵. These studies highlighted the existenceof unknown lethal processes involved in the elimination of irradiatedcells. Moreover, the increasing number of publications revealing theinfluence of cellular cannibalism and entosis in the control of tumorgrowth and in the elimination of metastatic cells urges one to followthe onset of this process of NCAD.

Considering the diversity of lethal stimuli that can potentiallyinitiate both non-cell autonomous and cell autonomous cell deathmodalities and the complexity of signaling pathways (involving (or not)caspases, cathepsins or granzymes) that control both processes, it wasdecided to simultaneously detect non-cell autonomous and cell autonomousdeath modalities elicited by IR. To determine whether after lethalinsults a cellular population may undergo simultaneously direct or/andby-stander-cell killing that may be executed in a cell-autonomous ornon-cell autonomous manner, a cell death profiling analysis was designedbased on the co-culture of HCT116 cells that have been labeled with5-chloromethylfluorescein diacetate (CMFDA, green) fluorescent vitalprobe and treated by ionizing radiations (y-rays) with isogenic HCT116cells that have been labeled with5-(and-6)-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine (CMTMR,red) fluorescent vital probe. After 24 hours of co-culture, treatedCMFDA⁺ cells, untreated CMTMR⁺ cells and the total (CMFDA⁺ cells andCMTMR⁺ cells) cell population were analyzed for phosphatidyl serine (PS)exposure, loss of plasma membrane integrity and DNA content tosimultaneously detect cell death induction of both treated cells and(untreated) neighboring cells. To characterize molecular mechanismsinvolved in the execution of detected cell death modalities, co-cultureswere performed in presence cell death modulators (such as ROCK1inhibitor (Y27632), pan-caspase inhibitor (ZVAD-fmk), caspase-1inhibitor (YVAD-fmk), necrostatin (NEC1), bafylomycine A1 (BafAl) andRoscovitine (Rosco)) that are respectively known to inhibit cellengulfment (a process that initiates entosis¹⁶, emperiptosis¹⁷ andcellular cannibalism¹⁶), proteolytic cleavage of caspase-3 or caspase-7(which contributes to the execution of apoptosis³⁹, mitoticdeath^(38, 40) or emperiposis¹⁷) or of caspase-1 (which is required forpyroptosis⁴¹), the activation of the pro-necroptotic kinase RIP1 kinase(RIPK1) which contributes to necroptosis⁴², the fusion betweenautophagosomes and lysosomes impairing thus the maturation of autophagicvacuoles during autophagy and autophagy-associated cell death⁴³) andfinally, the cyclin-dependent kinase 1 (Cdkl)-Cyclin B activity and theprogression through mitosis which is required for mitosis associateddeaths such mitotic death^(40, 44). The simultaneous detection of the PSexposure, loss of plasma membrane integrity, DNA content of cellularpartners combined with pharmacological inhibition of cell deathmodulators allowed us to detect during co-cultures through the use ofmultispectral imaging flow-cytometry the execution of at least 9 celldeath modalities (including apoptosis, mitotic death, pyroptosis,autophagic cell death, necrosis, necroptosis, entosis, emperitosis andcellular cannibalism) on targeted cells and on neighboring cells (FIG.1, A-B), thus discriminating non cell-autonomous deaths from cellautonomous deaths and direct cell killings from bystander lethaleffects. This methodology allowed one to define the cell death profilingelicited by IR.

Ionizing Radiation-Elicited Cell Death Profiling Highlights theInduction of Cell Death on Both Irradiated and Non-Irradiated CancerCells.

Despite the intensive biological and pharmaceutical research implementedto better characterize cellular and biochemical processes associatedwith anticancer treatments, lethal mechanisms responsible for thetherapeutic effects of radiotherapy, which is one of the most frequentanticancer treatment used in clinic, are still unknown. Lethal processes(such as apoptosis and mitotic catastrophe) that have been detected inresponse to ionizing radiation were not directly implicated in treatmentefficiency³⁵, suggesting that additional cell death modalities that arestill unknown may contribute to therapeutic effects of radiotherapy. Inthis context was determined the cell death profiling of irradiatedcancer cells. According to the above described methodology,CMFDA-labeled cells were irradiated or not with 4 grays, mixed after 24hours together at a 1:1 ratio with CMTMR-labeled cells, and cultured for24 hours in presence of each indicated inhibitors (Supplementary FIGS.1A-1E). Then, PS exposure, the membrane integrity and DNA content ofeach cellular partner were respectively determined using AnnexinV-BV786,DRAQ7 and Hoechst 33342 stainings. Despite no significant increase ofapoptotic and necrotic cell deaths (as revealed by the analysis ofAnnexinV⁺DRAQ7⁻ and DRAQ7⁺ cells) was observed on neighboring cellpopulation (CMTMR⁺ cells) (FIGS. 2A, 2B and 2D) and on untreated controlcell population (FIGS. 2A, 2D and 2E), a significant increase of theseboth types of death on total cell population (as revealed by consideringCMFDA⁺ and CMTMR⁺ cell population) (FIGS. 2A-2C) and on irradiated cellpopulation (CMFDA⁺ cells) (FIGS. 2B and 2E) was detected after 24-hoursof co-culture, demonstrating that the present methodology allows todetect cell death modalities of both non irradiated and irradiatedcells. It was also observed that the pan-caspase inhibitor Z-VAD-fmk andpharmacological cyclin-dependent kinase inhibitor roscovitine (Rosco)inhibited the exposure of PS on the outer plasma membrane of irradiatedCMFDA⁺ cells (FIG. 2E), confirming as previously published ^(45, 46)that irradiated CMFDA⁺ cells require both caspase activation andprogression through mitosis to die. In addition, the impairment ofautophagic flux with bafilomycin A1 (BafA1) increased the frequency ofdying cells (AnnexinV⁺DRAQ7⁻ and DRAQ7⁺ cells) in the total cellpopulation (CMFDA⁺ and CMTMR⁺ cells) (FIG. 2C), in non-irradiated CMTMR⁺cells (FIG. 2D) and in irradiated CMFDA⁺ cells (FIG. 2E), thus revealingthat autophagy is a survival cellular mechanism elicited by ionizingradiation that contributes to rescue both non-irradiated and irradiatedcells from death. In addition, the simultaneous analysis of theprogression of treated and untreated cells through their cell cyclesshowed that cell death inductions are associated with the escapement ofirradiated CMFDA⁺ cells from Gl arrest and led to their accumulations inS and G2/M phases (FIGS. 2F, 2G and 2J). No alteration of cell cycle isdetected on total or CMTMR⁺ cell populations, underlining that the cellcycle alterations are only detected on irradiated cells (FIGS. 2F, 2Gand 2I). These results, which were also confirmed by classicalflow-cytometry analysis (Supplementary FIGS. 2A-2B), demonstrated thatafter ionizing radiation, both irradiated and non-irradiated cancercells undergo caspase-1 dependent cell death. Altogether, these resultshighlighted the ability anticancer agents to simultaneously eliminatecancer cells through direct cell killing and bystander effects.

Ionizing Radiation-Elicited Cell Death Profiling Also Reveals theInduction of Non-Cell-Autonomous Death Modalities.

In parallel, in the same co-culture was determined the ability ofirradiated CMFDA⁺ cells to engulf or to invade neighboring cells, twocellular processes required for the induction cellularcannibalism-associated cell deaths (such as cellular cannibalism,emperitosis or phagoptosis) or cell-in-cell invasion-elicited celldeaths (such as entosis). Multispectral imaging flow-cytometry analysisrevealed that gamma-irradiated CMFDA⁺ cells triggered the engulfment ofneighboring cells (as revealed by the internalization of “target” CMTMR⁺cells by gamma-irradiated CMFDA⁺ cells (FIGS. 3A and 3B)). This processthat was not affected by the pan-caspase inhibitor Z-VAD-fmk isrepressed by the inhibitor of ROCK1 (Y27632) (FIG. 3B), thus revealingthat the detected cell-in-cell internalization is distinct fromphagocytic uptake of apoptotic cells and requires ROCK1 activity tooccur. Cannibalistic activity of gamma-irradiated cells was alsoconfirmed by confocal microscopy (FIG. 3D-3F). Interestingly, cell deathprofiling analysis also allowed to distinguish live cell engulfment fromthe phagocytosis of apoptotic CMFDA⁺ cells by lived CMTMR⁺ cells that isconsecutive to the induction of apoptosis by the treatment withbafylomycin A1 (FIG. 3B). Then, the cell fates of engulfed CMTMR⁺ cellsand irradiated engulfing CMFDA⁺ cells were evaluated. It was observedthat almost all engulfed CMTMR⁺ cells exhibited signs of cellulardegradation (as revealed by the DNA content loss of internalized cellsdetected with multispectral imaging flow-cytometry (FIG. 3A) andconfocal microscopy (FIGS. 3D and 3E)). This process is significantlyreduced in presence of the pan-caspase inhibitor Z-VAD-fmk and thecaspase-1 inhibitor YVAD-fmk, revealing that IR-mediated death ofengulfed cells requires caspases to occur and may be executed throughcaspase-1 dependent apoptosis, that is also known as pyroptosis. Inaddition, it was also revealed that 90% of cannibal cells did not exposePS or exhibit loss of the integrity of their plasma membrane(Supplementary FIG. 2C) underlining that after IR-mediated cellengulfment, the internalized cells are precipitated to death withoutmodulating the viability of cannibal cells. Altogether, these resultsdemonstrated that ionizing radiation simultaneously eliminates cancercells through combined effects of direct cell killing, bystander lethaleffect and cellular cannibalism-associated cell death. These resultshighlight the urgent need to simultaneously measure the induction ofnon-cell autonomous and cell autonomous death subroutines during lethalprocesses.

Chemical Library Screening Leads to the Identification of IR-MediatedCellular Cannibalism Enhancers.

Then, the screening of a library of chemical compounds was developed inorder to identify compounds able to induce IR-mediated cellularcannibalism. Thus, HCT116 cells that have been treated with a radiationdose of 8 Gray were stained either with orange CMTMR cell tracker orgreen CMFDA cell tracker and cultivated in presence of 10 μM of chemicalcompounds. After 24 hours of culture, cells have been stained withnuclear dye (5 μg/ml of Hoechst 33342 during 1h at 37° C.) and analyzedusing the FlowSight® Imaging Flow Cytometer for cellular cannibalism.Each of these compounds were classified according to their Z-score⁴⁷ andidentified respectively 13 and 11 candidate compounds (FIGS. 4A, 4B, 4Fand 4G). Then, the identified compounds were validated by combining flowcytometry approaches (in order to eliminate cytotoxic compounds bysimultaneously monitoring the depolarization of the internalmitochondrial membrane and the plasma membrane permeability of thetreated cells) with confocal microscopy approaches (to confirm themodulation of IR-induced cellular cannibalism by candidate compounds)(FIGS. 4C-E and 4H-J). After the completion of three independentexperiments, chemical compounds with no effect (such as Fenbendazol orCarbimazole) or exhibiting high cytotoxicity (such as RN-1-183) havebeen eliminated. Finally, 16 chemical compounds that are able to enhancethe capacity of IR to trigger cellular cannibalism were identified(FIGS. 4E and 4J, Tables 1&2).

The Combination of IRCCE with IR Induces Efficient Antitumor Immunity.

Considering that immunogenic cell death (ICD) inducers were identified(such as Digoxin, Digitoxin, Lanatoside C or Doxorubicinhydrochloride)^(3, 4, 48-51)) as IRCCE, the ability of these compoundsto induce an antitumor immunity after radiotherapy was evaluated. First,a preclinical approaches was developed to study the immunologicaleffects of IRCCE on two mouse models of carcinoma (colon CT26 carcinomaand fibrosarcoma MCA205). Initially, the ability of the combination ofIRCCE+IR to trigger a specific anti-tumor immune response wasappreciated using immunocompetent mice through anti-tumor vaccinationassays. According to previously published studies⁵², the injection ofcancer cells succumbing to an immunogenic cell death (ICD) intoimmunocompetent mice must elicit a protective immune response that isspecific for tumor antigens. Thus, 3×10⁵ MCA205 cells were first treatedwith 10 μM of SG6163F for 24 hours. Then, treated cells were inoculatedsubcutaneously in 200 μl PBS into the lower flank of 8-week-old femaleC57BL/6 mice. One week later, 3×10⁴ untreated control cells wereinoculated into the contralateral flank of mice. Tumors were evaluatedweekly using a common caliper. Animals bearing tumors that exceeded20-25% body mass were euthanatized. It was observed that mice treatedwith IR or SG6163F alone are not able to induce a significant increaseof cell death in vitro (FIG. 5A). In fact, they did not exhibit aprotective response after the inoculation of untreated cells, revealingthat the dose of radiation and the concentration of SG6163F used werenot sufficient to stimulate an anticancer immune response (FIG. 5B).These results were consistent with previously published resultsdemonstrating that a protective response is only detected when cancercells were exposed to cytotoxic anticancer treatment in vitro until 70%of the cells expose phosphatidylserine on the outer leaflet of theplasma membrane⁴⁸. Surprisingly, it was observed that the combinedtreatment of MCA205 cells with 8 Gy of IR and 10 μM of SG6163F induced aprotective response in 3 of 5 mice that have been injected (FIG. 5B).These results revealed for the first time that the combination ofIRCCE+IR may elicit an IR-mediated anticancer immune response in absenceof a significant increase of death of treated and irradiated cells (FIG.5A), suggesting that cellular cannibalism or cellularcannibalism-associated signaling pathways may contribute to theinduction of tumor immunogenicity. Then, 3×10⁶ CT26 cells wereirradiated with 8 Gy in presence of 10 μM of SG6163F (FIGS. 5E and 5F),VP331 (FIGS. 5I and 5J), Minaprine dihydrochloride (FIGS. 5M and 5N) orLOPA87 (FIGS. 5Q and 5R) for 24 hours. Then, cells were inoculated asprevisouly described subcutaneously in 200 l PBS into the lower flank of8-week-old female BALB/c mice. One week later, 5×10⁵ untreated controlcells were inoculated into the contralateral flank of mice and tumorswere evaluated weekly using a common caliper. As previously mentioned,animals bearing tumors that exceeded 20-25% body mass were euthanatized.The percentage of dying cells in each condition was evaluated bydetermining with DiOC(6)3/IP staining (FIGS. 5E, 5I, 5M and 5Q).Finally, the ability of these compounds to repress the growth of cancercells that have been injected 7 days after injection of irradiated andtreated cancer cells was appreciated. It was observed a significantincrease in the frequency of mice showing a protective response afterinjection of cancer cells that have been treated with IR and chemicalcompounds (FIGS. 5H, 5L, 5P and 5T).

A second anti-tumor vaccination assay was done on CT26 mouse models ofcarcinoma as previously described. Briefly, CT26 cells were irradiatedwith 8 Gy in presence of 10 μM of VP331 (FIG. 6A, 6F, 6K), Minaprinedihydrochloride (FIG. 6B, 6G, 6L), LOPA87 (FIG. 6C, 6H, 6M), SG6163F(FIG. 6D, 6I, 6N), or Azaguanine-8 (8-aza) (FIG. 6E, 6J, 6O) for 24hours. Then, cells were inoculated as previously described intoimmunocompetent BALB/c mice. Finally, the ability of these compounds torepress the growth of cancer cells that have been injected 7 days afterinjection of irradiated and treated cancer cells was appreciated. Theprevious results were confirmed. Significant increases in the frequencyof mice showing a protective response after injection of cancer cellsthat have been treated with IR and chemical compounds was observed(FIGS. 6P and 6Q). Interestingly, minaprine dihydrochloride (FIGS. 6B,6G, 6L, 6P and 6Q) and azaguanine-8 (FIGS. 6E, 6J, 6O, 6P and 6Q) aloneinduce an increase of protective response after injection of cancercells.

Altogether, these results revealed the ability of chemical compoundsidentified with the present platform to induce a protective anticancerimmune response.

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1. An in vitro method of identifying a compound that may potentiate aradiotherapy treatment in a subject, said method comprising the stepsof: (a) adding a candidate compound to a tumor cell culture, (b)treating said tumor cell culture with a radiation dose of 1-20 Gray, (c)measuring the irradiation-mediated cellular cannibalism occurring insaid cell culture, wherein a compound that enhances irradiation-mediatedcellular cannibalism as compared to control levels will be able topotentiate a radiotherapy treatment.
 2. In vitro use of the compoundshighlighted in Tables 1 & 2 and analogs thereof, for enhancingIR-mediated cellular cannibalism in cancer cells.
 3. Compounds disclosedin Tables 1 & 2 or their analogs, or any pharmaceutical compositioncontaining same, for their use as enhancers of IR-mediated cellularcannibalism in patients suffering from cancer.
 4. Enhancers ofIR-mediated cellular cannibalism, for their use for enhancing tumorimmunogenicity in subjects that will receive or that have received aradiotherapy treatment.
 5. Enhancers of IR-mediated cellularcannibalism, for their use for inducing a significant protectiveanticancer immune response in subjects that will receive or that havereceived a radiotherapy treatment.
 6. Enhancers of IR-mediated cellularcannibalism, for their use for potentiating a radiotherapy treatment ina subject in need thereof.
 7. Enhancers of IR-mediated cellularcannibalism, for their use for treating cancer, in conjunction withradiotherapy, in a subject in need thereof.
 8. Enhancers for useaccording to claims 4-7, wherein said enhancers are disclosed in Tables1 & 2 or their analogs, preferably chosen in the group consisting of:Mebhydroline 1,5-napthalene disulfonate salt, Flurbiprofen, Minaprinedihydrochloride, Myricetin, Digoxin, Digitoxin, Lanatoside, LOPA87,VP331, RN-1-026, SG6163F, VP450, and VP43.
 9. Enhancers for useaccording to claims 4-7, wherein said enhancers are chosen in the groupconsisting of: Minaprine dihydrochloride, LOPA87, VP331, and SG6163F.10. Enhancers for use according to claims 4-9, wherein said subject orpatient suffers from brain cancer (e.g., glioma), gastric cancer,head-and-neck cancer, pancreatic cancer, non-small cell lung cancer,small cell lung cancer, prostate cancer, colon cancer, non-Hodgkin'slymphoma, sarcoma, testicular cancer, acute non-lymphocytic leukemia orbreast cancer.
 11. Enhancers for use according to claims 4-10, whereinthey are administered prior to a radiotherapy treatment, preferably 24hours prior to the radiotherapy treatment.
 12. Enhancers for useaccording to claims 4-10, wherein they are administered after aradiotherapy treatment.
 13. Enhancers for use according to claims 4-10,wherein they are administered concomitantly to a radiotherapy treatment.14. Minaprine dihydrochloride for use for preventing or treating cancerin a subject in need thereof.
 15. Minaprine dihydrochloride for useaccording to claim 14, for treating cancer in conjunction with aradiotherapy treatment.
 16. Minaprine dihydrochloride for use accordingto claims 14 or 15, wherein said subject suffers from brain cancer(e.g., glioma), gastric cancer, head-and-neck cancer, pancreatic cancer,non-small cell lung cancer, small cell lung cancer, prostate cancer,colon cancer, non-Hodgkin's lymphoma, sarcoma, testicular cancer, acutenon-lymphocytic leukemia or breast cancer.